Device and method for detecting blood group antigens by means of an incomplete antibody

ABSTRACT

A device for determining a cellular-bound analyte in a liquid sample includes a separation matrix with at least one indicator zone. The indicator zone includes a first antibody directed against the cellular-bound analyte or a fragment thereof and a binding element directed against the first antibody. The first antibody is an incomplete antibody. The separation matrix can be designed in the form of the membrane of a lateral flow assay device or as a gel matrix. For example, the device can include a membrane with a charging zone for applying the liquid sample, at least one indicator zone which can interact with the cellular-bound analyte, and at least one absorption region, which absorbs the liquid after passing the indicator zone. The indicator zone lies between the charging zone and the absorption region. The indicator zone can include an antibody directed against the cellular-bound analyte or a fragment thereof and a binding element directed against the first antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. national stage entry under 35 U.S.C. §371 of PCT International Application No. PCT/EP2015/001067, which wasfiled on May 23, 2015, which claims priority to German PatentApplication No. 10 2014 007 851.5, which was filed on May 26, 2014, thedisclosures of each of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The invention relates to devices and methods for determining blood groupantigens by means of an incomplete antibody, in particular forsimultaneously determining blood group antigens.

PRIOR ART

In blood group serological diagnostics, parameters which are importantespecially in connection with transfusions or haemolytic disease of thenewborn are generally detected. This includes inter alia the detectionof antigens on the surface of erythrocytes which are characteristic forthe blood groups. Further important antigen systems are also found onthrombocytes, granulocytes and lymphocytes, which likewise play a rolein transfusion and/or transplantation.

It is known that, in order to determine the blood group antigens, theerythrocytes of the person to be tested (donor or recipient) are broughttogether with reagents which contain blood-group-specific antibodies.The tests are usually liquid tests, in which a test batch is produced bymixing an erythrocyte-containing sample with a sample containingantibodies directed against a specific blood group feature. The testbatch is then incubated for a defined period of time and under definedconditions and, when the incubation is complete or directly after acentrifugation step, the batch is checked either visually or by opticalmethods for possible agglutination or adsorption of the erythrocytes.The prevailing end-point measurement in blood group serology continuesto be haemagglutination. Direct agglutinating antibodies are alsoreferred to as complete antibodies in blood group serology. Antibodieswhich cannot agglutinate erythrocytes directly are analogously referredto in blood group serology as incomplete antibodies.

The simultaneous determination of blood group antigens using a lateralflow test format is known from WO 2005/005991. The examples of WO2005/005991 disclose the determination of blood group antigens by meansof IgM antibodies, which are complete antibodies and lead directly tohaemagglutination. The WO specification does not, however, disclose thedetermination of blood group antigens using an incomplete antibody withthe aid of a lateral flow test.

Lateral flow tests are widely used nowadays as rapid tests, for exampleas pregnancy tests, for determining infection markers or as a drugscreen. A lateral flow test arrangement consists of a solid carrier towhich there is applied an work zone for the sample to be tested, aseparating membrane on which binding elements, for example captureantibodies or antigens, are bound and on which binding reactions can bedetected, and an absorbent absorption region which allows the sample tobe tested to flow through the separating membrane.

Test membranes of conventional lateral flow tests are generallydescribed having chromatography-like separation. The analyte in thesample binds specifically to the binding elements fixed in a membrane,which are generally arranged as indicator zones in bands located onebehind the other or one above the other. The binding complex is madevisible by indicator particles, which are generally already present inthe arrangement in dried form in a conjugate release pad. The conjugaterelease pad is typically disposed between the work zone and themembrane. The pre-coated coloured indicator particles are coated, forexample, with an antibody directed against the analyte being sought.

The most important blood group features which nowadays must routinely beclarified in pre-transfusion tests on the patient and on donors are: A,B, D, C, E, c, e, Cw, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s, P1, Lea,Leb, Kpa, Kpb, Lua, Lub. The antigens or antigen epitopes to be testedare, by way of example, those of the ABO blood group system, of the Rh,Kell, Lewis-Hh, Duffy-Kidd, MNS, Lutheran and P system, of the bloodgroup systems Diego, Yt, Scianna, Dombrock, Colton, Chido/Rodgers,Gerbich, Cromer, Knops, Landsteiner-Wiener, Xg, Kx, Indian, Ok, Raph,John Milton Hagen, Langereis, Sid, FORS, JR and/or LAN, in particularA1, A2, AB, B, D, C, c, E, e, Cw, K, k, M, N, S, s, Jka, Jkb, Fya, Fyb,Kpa, Kpb, Jsa, Jsb, Lea, Leb, Lua, Lub, P1, I, H, Xga, U, Vw, Wra, Lan,Vel, Dia and/or Mia.

Because of their negative net surface charge and the zeta potentialthereby exerted, erythrocytes have a natural statistical minimumdistance of approximately 300 angstroms between the cells. That minimumdistance can be bridged by antibodies of the IgM class in physiologicalmedium because of the molecule size, but naturally not by antibodies ofthe IgG class. This means that, as a rule, only IgM-class antibodies areavailable in blood group serology for a direct end-point measurement byhaemagglutination. Direct agglutinating antibodies are also referred toas complete antibodies in blood group serology (most IgM antibodies arecomplete antibodies).

According to the current prior art, blood groups generally cannot bedetected by means of IgG antibodies by direct haemagglutination.Antibodies which cannot agglutinate erythrocytes directly are referredto analogously in blood group serology as incomplete antibodies (mostIgG antibodies are incomplete antibodies).

This leads to the situation that it is necessary to work with so-calleddifferent phases and reaction times and temperatures according towhether (monoclonal) IgMs or, on the other hand, monoclonal IgGs orpolyclonal antibodies are available for detection for a particular bloodgroup property, which makes harmonised or homologised working proceduresmore difficult.

Provided that IgM antibodies are available, direct determination withhaemagglutination as the end point is frequently possible without theadmixture of further antibodies or intensifiers or proteolytic enzymesand without incubation (immediate spin). With the widely used geltechnique, incubation is not necessary for the performance of such atest; the reaction mixture comprising the erythrocytes to be tested andthe antibody reagent simply has to be pipetted into the work zones ofthe gel card and centrifuged for 9-10 minutes in a neutral physiologicalmedium, that is to say a physiological medium which does not containantibodies (for example DG Gel Neutral Card from Diagnostic Grifols). Inanother variant of the same technique, blood-group-specific antibodiesof the IgM class have already been introduced into the gel matrix. Theerythrocytes to be tested then simply have to be pipetted into the workzones of the gel card (for example DG Gel ABO RH (2D) from DiagnosticGrifols).

If the antibody available for determining a particular blood groupfeature does not belong to the IgM class, a technique/phase change isrequired in order to make haemagglutination possible as the end point.This is the case, for example, for the following of the above-mentionedfeatures, for which no commercially available IgM antibodies areavailable according to the current prior art: k, Fya, Kpa, Kpb and Lua.The following further features are likewise of interest, such as Dia,Jsa, Jsb, Coa, Cob, Wra, Xga. Commercial monoclonal IgM antibodies arenot available for detecting any of these antigens.

Since IgG antibodies are generally not capable of overcoming thedistance that is present between two erythrocytes due to naturalrepulsion, reaction with an antigen-specific IgG antibody can achieveonly sensitisation (that is to say antibody binding, but nothaemagglutination, therefore no diagnostic end point) of the cells thatare positive for the particular antigen, without the visible end pointof haemagglutination, which in turn is necessary for simple visualdiagnostic detection: If, for example, an erythrocyte which carries theblood group feature Duffy a (Fya) is incubated with an anti-Fya antibodyof the IgG class, the antibody-antigen reaction (sensitisation) occurs,but this does not lead to the visible end point of haemagglutination. Inorder to achieve this, the sensitised cells must additionally beincubated with a class-specific antibody (in the present case anti-IgG),with the aid of which the cells sensitised with IgG antibodies can bebridged and the end point of haemagglutination can be produced (indirectCoombs test). The gel technique, which is widely used for this purpose,requires an incubation time of 10-15 minutes at 37° C. for this test,with subsequent centrifugation for 9-10 minutes in an anti-humanglobulin or Coombs card (for example DG Gel Coombs Card from DiagnosticGrifols).

In the tube technique, which is likewise widely used, incubation priorto centrifugation for approximately 20 seconds is not necessary in thecase of immediate spin. For the indirect Coombs test, incubation isfirst carried out for from 15 to 60 minutes at 37° C. with theblood-group-specific incomplete antibody, following which a plurality ofwashing steps are required before the anti-human globulin reagent isadded and centrifugation is then carried out for 20 seconds.

There is therefore a need for a device and a method for determiningcell-bound analytes, in particular blood group antigens, for which nostandardised IgM antibodies, in particular no commercially available IgMantibodies, are available. There is further a need for a device and amethod for simultaneously determining at least two cell-bound analytes,wherein a standardised IgM antibody such as a commercially availableantibody is available for only one of the two cell-bound analytes, sothat the determination of both analytes in the prior art requires aphase or technique change.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda device for determining a cell-bound analyte in a liquid sample,comprising a separating matrix having at least one indicator zone,characterised in that the indicator zone comprises a first antibodydirected against the cell-bound analyte, or a fragment thereof, and abinding element directed against the first antibody, the first antibodybeing an incomplete antibody.

According to a preferred embodiment, the device comprises a membrane (2)having an work zone (5) for application of the liquid sample, at leastone indicator zone which is able to interact with the cell-boundanalyte, and at least one absorption region (3) which absorbs the liquidafter it has passed through the indicator zone, the indicator zone beingsituated between the work zone (5) and an absorption region (3),characterised in that the indicator zone comprises a first antibodydirected against the cell-bound analyte, or a fragment thereof, and abinding element directed against the first antibody, the first antibodybeing an incomplete antibody.

According to a further preferred embodiment, the device contains tubesfilled with gel material. The gel technique is used to determine theagglutination reaction of erythrocytes. The gel column acts as a filterwhich slows or stops the migration of the agglutinated erythrocytesrelative to non-agglutinated erythrocytes and thereby effectsseparation. According to the invention, the indicator zone of the gelcontains an antibody directed against the cell-bound analyte, or afragment thereof, and a binding element directed against the firstantibody, the first antibody being an incomplete antibody.

According to a second aspect of the present invention, there is provideda device for simultaneously determining a first and a second cell-boundanalyte in a liquid sample, comprising a membrane (2) having an workzone (5) for application of the liquid sample, at least two indicatorzones which are able to interact with the cell-bound analytes, and atleast one absorption region (3) which absorbs the liquid after it haspassed through the indicator zone, the indicator zones being situatedbetween the work zone (5) and the at least one absorption region (3),characterised in that (i) the first indicator zone comprises a firstantibody directed against the first cell-bound analyte, or a fragmentthereof, and a binding element directed against the first antibody, thefirst antibody being an incomplete antibody, and (ii) the secondindicator zone (a) comprises a first antibody directed against thesecond cell-bound analyte, the first antibody being a complete antibody;or (b) comprises a first antibody directed against the second cell-boundanalyte, the first antibody being incomplete, and a binding elementdirected against that antibody.

Surprisingly, the inventors of the present application have found that,by applying a first incomplete antibody and a second antibody directedagainst the first antibody together in an indicator zone, it is possibleto configure a device having a separating matrix, preferably in the formof the membrane of a lateral flow test device or as a gel matrix, insuch a manner that it is possible to determine a cell-bound analyte bymeans of an incomplete antibody as the first antibody. As a result, itis possible for the first time to determine a cell-bound analyte, forwhich no standardised, such as commercially available, antibodies of theIgM type are available, using a separating matrix such as a lateral flowtest device. This results in a considerable shortening of the timerequired for determining such analytes, which previously could generallybe determined only by means of the indirect Coombs test, which requiresan additional incubation step. The procedure according to the inventionis also surprising for the skilled person because he would have assumedthat, for example when using anti-IgG molecules as the second antibody,these were neutralised by the non-analyte-specific IgG molecules presentin a high concentration in whole blood.

It was therefore not possible in the prior art to determine two bloodgroup antigens simultaneously (that is to say in a single lateral flowdevice or in a single gel card having a plurality of gel tubes fordetermining a plurality of parameters), the first being determined by anIgG antibody and the second by an IgM antibody, without a technique orphase change thereby being required. The present invention thereforeoffers the advantage of simultaneous determination using a singlelateral flow set-up, which requires only a single homogeneous methodstep without different media and different incubations.

According to a third aspect of the present invention, there is provideda method for producing the above devices, comprising

-   -   applying a first antibody directed against a cell-bound analyte,        or a fragment thereof, and a second antibody directed against        the first antibody, or a fragment thereof, wherein the first        antibody is an incomplete antibody.

According to a fourth aspect of the present invention, there is provideda method for determining at least one cell-bound analyte, comprising

-   -   applying a first antibody directed against a cell-bound analyte,        or a fragment thereof, and a binding element directed against        the first antibody in the indicator zone, wherein the first        antibody is an incomplete antibody.

According to a fifth aspect of the invention, there is provided the useof the devices according to the invention for analysing blood, inparticular for determining blood group antigens or antigen epitopes.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In connection with the present invention, the following expressions areto have the meanings given below:

The expression “complete antibody” means an antibody which leads to theagglutination of erythrocytes in the physiological saline medium.Complete antibodies include IgM antibodies or fragments thereof,provided the fragments are still capable of agglutination. The IgMantibodies can be monoclonal or polyclonal.

The expression “incomplete antibody” means an antibody which, whenincubated with erythrocytes, does not lead to the agglutination thereof.Incomplete antibodies include IgG antibodies. IgA antibodies, IgDantibodies and IgE antibodies including their subclasses or antibodyfragments, provided the fragments are still capable of binding a secondantibody directed against the entire antibody. These antibodies can bemonoclonal or polyclonal. Methods of producing antibodies of the variousclasses are known to the skilled person.

The expression “cell-bound analyte” means any molecule that is naturallybound to the surface of a cell, preferably of a human cell, inparticular of an erythrocyte. They include, for example, receptors orblood group antigens, blood group antigens being preferred.

The expression “blood group antigen” includes antigens of the ABO bloodgroup system, of the Rh. Kell. Lewis-Hh, Duffy-Kidd. MNS. Lutheran and Psystem, of the blood group systems Diego, Yt, Scianna, Dombrock, Colton.Chido/Rogers, Gerbich, Cromer, Knops, Landsteiner-Wiener, Xg, Kx,Indian, Ok, Raph, John Milton Hagen, Langereis, Sid, FORS, JR and/orLAN, in particular A1, A2, AB, B, D, C, c, E, e, Cw, K, k, M, N, S, s,Jka, Jkb, Fya, Fyb, Kpa, Kpb, Jsa, Jsb, Lea, Leb, Lua, Lub, P1, I, H,Xga, U, Vw, Wra, Lan, Vel, Dia and/or Mia.

Production of the Lateral Flow Device

A method that is suitable in principle for producing a lateral flowdevice is described in DE 10330982 A1 and WO 2005/005986, but it isaltered as indicated hereinbelow. The disclosure of DE 10330982 A1 andWO 2005/005986 is incorporated herein by reference.

The method for producing a device according to the invention comprises

-   -   applying a first antibody directed against a cell-bound analyte,        or a fragment thereof, and a binding element directed against        the first antibody in the indicator zone, wherein the first        antibody is an incomplete antibody.

The first antibody directed against a cell-bound analyte and the bindingelement directed against the first antibody can be applied to themembrane in the region of the indicator zone either together orseparately from one another. Where they are applied separately from oneanother, it is preferred that a drying step takes place after theapplication of the first antibody, before the binding element isapplied. The concentration of the first antibody is determinedempirically and depends on the affinity for the cell-bound analyte. Theconcentration of the binding element can be optimised by test series onthe basis of the concentration of the first antibody and the propertiesthereof.

The analyte to be determined is preferably a blood group antigen. Thefirst antibody is particularly preferably directed against a cell-boundanalyte selected from the blood group antigens A, B, AB, D, C, E, c, e,Cw, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s, P1, Kpa, Kpb, Lua, Lub, Lea,Leb, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra and Xga, particularly preferablyagainst k, S. Fya, Kpa, Kpb, Lua, Lea, Leb, Mia, Lua, Lub, Dia, Jsa,Jsb, Coa, Cob, Wra and Xga. The first antibody is an incomplete antibodyin this case, preferably an IgG or IgA antibody, particularly preferablyan IgG antibody. For example, the following antibodies can be used:anti-Fya: clone P3TIM (Merck Millipore, VL); anti-S: clone P3S13JS123(Diagast, ref. 78007); anti-k: clone P3A118OL67 (Merck Millipore, FA);and anti-D: clone ESD-1 (Alba Bioscience).

The binding element is preferably selected from an antibody directedagainst the first antibody, or a fragment thereof, and a lectin or afragment thereof. The antibody directed against the first antibody isparticularly preferably an anti-IgG antibody. Anti-IgG antibodies arecommercially available, particularly preferred are the clone MS-278(Merck Millipore) and as a polyclonal antibody, for example, mono-typeor anti-IgG anti-human globulin (Medion Grifols Diagnostics). Where thefirst antibody is an IgA antibody, the second antibody is an anti-IgAantibody. Anti-IgA antibodies are commercially available. The anti-IgGor anti-IgA antibodies can be of the IgM or IgG type, a monoclonalanti-IgG of the IgM class being preferred. Preferred lectins are proteinA and protein G.

Where a device for simultaneously determining a first and a secondcell-bound analyte according to the second aspect of the presentinvention is to be produced. (i) the first indicator zone comprises afirst antibody directed against the first cell-bound analyte, or afragment thereof, and a binding element directed against the firstantibody, wherein the first antibody is an incomplete antibody, and

(ii) the second indicator zone comprises (a) a first antibody directedagainst the second cell-bound analyte, wherein the first antibody is acomplete antibody; or (b) a first antibody directed against the secondcell-bound analyte, wherein the first antibody is incomplete, and abinding element directed against that antibody.

The first cell-bound analyte is preferably selected from the blood groupantigens k, Fya, Kpa, Kpb, Lua, Lub, Mia, Dia, Jsa, Jsb, Coa, Cob, Wra,Xga and S and the second cell-bound analyte is preferably selected fromA, B, AB, C, D, E, c, e, Cw, K, Lea, Leb, Jka, Jkb, Fyb, P1 and s.

The first antibody directed against the second cell-bound analyteaccording to alternative (a) is a complete antibody, in particular anIgM antibody, which leads directly to haemagglutination. The firstantibody directed against the second cell-bound analyte according toalternative (b) is an incomplete antibody, preferably the first antibodyaccording to alternative (b) is an IgG or IgA antibody. The bindingelement directed against the first antibody according to alternative (b)permits determination by haemagglutination. The binding element ispreferably an IgG or IgM antibody. Alternatively, a lectin such asprotein A or protein G may also be used.

The membrane of the device used according to the invention is a porousmembrane. Preferred membrane materials are, for example, nitrocellulose(for example UniSart from Sartorius. HiFlow from Millipore, Whatman,AE99 or FF85/100 from Whatman Schleicher & Schuell), polyethylene(Lateral Flo from Porex Corporation) or nylon (Novylon from CUNO). Themembrane preferably has as large a pore size as possible, since a highporosity of the membrane facilitates the penetration in particular ofcell components of the sample to be determined, for example oferythrocytes, into the porous structure. The use of absorbent membranesis particularly advantageous. However, the device according to theinvention is not limited to those properties. Preference is given to anymembranes having a high capillary flow rate, where the capillary flowrate is the time [s] required for a dye solution to cover a distance of40 mm on a given membrane. Membranes whose capillary flow rate is <100are particularly preferred.

In a preferred embodiment of the invention, a sealing element isarranged on the porous membrane downstream, in relation to the directionof flow, of the work zone of the device according to the invention. Two-or three-dimensional sealing elements are used, which are placed on theporous membrane and with which a sample work zone separated from theremainder of the surface of the porous membrane is created. According tothe invention, the sealing element acts primarily as a liquid barrierand permits the directional distribution of sample liquid and testreagents into the porous membrane. According to the invention, thesealing element further seals off the sample work zone in order toprevent liquid from undesirably entering the other regions of thelateral flow device.

Preferred embodiments of the sealing element are the web or trough orfunnel shape. The sealing element is cut the material used to producethe sealing element. In the case of the funnel or trough shape, thesealing element is provided with an inner opening, preferred variants ofwhich are round, square or rectangular shapes which, in the case of thefunnel shape, taper towards the underside (membrane contact side) of thesealing element. Preferred materials for the sealing element arematerials which do not absorb water (hydrophobic). In a particularembodiment, the materials are coated on one side with an adhesive film,for example a pressure-sensitive or self-adhesive acrylate adhesive. Thesealing element can thus be bonded directly to the surface of the porousmembrane. Alternatively, the sealing element can be connected, forexample adhesively bonded, to the lateral flow casing, the lateral flowcasing in this embodiment pressing the sealing element on the surface ofthe porous membrane and the functions of the sealing element therebybeing achieved.

Preferred materials for forming two-dimensional sealing elements are anyform of adhesive tape or adhesive foil (for example Tesa 4124 fromBeiersdorf AG, ARcare 7815 from Adhesives Research). Preferred materialsfor forming three-dimensional sealing elements are flexible, closed-poreelastomer materials or flexible silicone materials with differentmaterial thicknesses, preferably 3-5 mm (for example EPDM140 cellularrubber from Pitzner, silicone rubber or solid rubber, hardness 40 deg.or less, from Castan).

In a further preferred embodiment, multiple scaling elements consistingof one piece with, for example, 20 individual cavities (trough shape)are arranged on one membrane.

As a result of this design, the device according to the invention iscapable of absorbing liquid samples which contain cells, such as wholeblood, without thereby filtering off the cells. Furthermore, the sealingelement allows large sample volumes to be applied to the porous membrane(work zone) without flooding it. The scaling element thus supports theuse of the absorbing properties of the porous membrane. Furthermore, thesealing element ensures a directional sample flow. The device accordingto the invention can, however, function well with or without a sealingelement.

For the absorption region (absorption pad) of the device according tothe invention, preference is given to mechanically stable materials,preferably having water absorption capacities of 20-30 g/100 cm² (forexample Millipore). The contact between the absorption pad and thelateral flow membrane of the device according to the invention isestablished by pressure and overlapping with the porous membrane.Precise positioning of the absorption pad on the membrane is achieved byadhesively bonding the absorption pad to the backing sheet carrying thelateral flow membrane.

In a further embodiment, the components of the device according to theinvention are applied to a substrate or backing sheet for the purposesof mechanical strengthening. The device according to the invention can,however, function with or without a backing sheet. Preference is givento mechanically stable materials which do not absorb water, preferablywith material thicknesses of 100 μm or more, which are coated on oneside or on both sides with an adhesive film, for example apressure-sensitive or self-adhesive acrylate adhesive (for example0.005″ polyester W/GL-187, G&L). The porous membrane and the absorptionpad are fixed to the backing sheet. In the case of a backing sheet thatis adhesive on both sides, the adhesive second side is used for fixingthe stack to further surfaces, for example inside the lateral flowcasing.

In a further embodiment, the device according to the invention, eitherwith or without a backing sheet to which the components of the deviceaccording to the invention are applied, is integrated in a casing, themembrane components thereby being pressed against one another and thecasing supporting the function of the sealing element. The deviceaccording to the invention can, however, function equally as well withor without a casing in this case.

Determination Methods

The method is carried out by applying a liquid sample. The liquid samplepreferably consists of blood or constituents of blood, particularlypreferably of whole blood, erythrocyte concentrate, coagulated blood ortest liquid, such as control blood. The sample may be diluted with abuffer in this case before it is applied.

The invention will be explained in greater detail below by means offigures and examples, without being limited thereto.

FIG. 1 is, by way of example, a perspective view of a device accordingto the invention for lateral flow tests for simultaneously determiningblood group antigens. In the present example, the device consists of abacking sheet 1, the porous membrane 2, the absorption pad 3 and thesealing element 4, which is two-dimensional in web form orthree-dimensional in trough form. The porous membrane 2 is thereby fixedto the backing sheet 1 provided with a pressure-sensitive orself-adhesive acrylate adhesive. The absorption pad 3 is likewise fixedto the backing sheet 1, some of the absorption pad 3 overlapping theporous membrane 2. The sealing element 4 fixed to the upper side of theporous membrane 2 separates the work zone 5 from the remainder of themembrane surface and permits the directional distribution of sampleliquid and test reagents into the porous membrane 2. The indicator zoneregion 6 is arranged between the work zone 5 and the region of theporous membrane 2 that is in contact with the absorption pad 3.

FIG. 2 shows a successful simultaneous determination of the blood groupantigens Jka, Jkb, Fya, Fyb, S, s, k and P1. The donor isJka−Jkb+Fya−Fyb+S−s+k+P1+. The sample was hereby applied to the workzone situated in the middle. The sample flows through both the indicatorzones situated to the left of the work zone and the indicator zonessituated to the right of the work zone.

FIG. 3 shows a comparison of, on the one hand, the method according tothe invention with a first IgG antibody directed against the blood groupantigens D, Fya and k and a second anti-IgG antibody directed againstthe first antibody, and, on the other hand, a comparative method withoutthe second antibody. On the right-hand side, anti-IgG is applied threetimes as a further negative control. FIG. 3a shows the dispensing planused. FIG. 3b to 3e show the experimental results which were obtainedusing samples from different donors.

EXAMPLES Example 1: Blood Group Determination Production of the TestStrips:

The test strips consist of an work zone located in the middle of themembrane, as well as two indicator zone regions and two absorptionregions at equal distances on both sides of the central work zone.Membranes of the Millipore HiFlow Plus 065 type are trimmed in strips toa size of 19×48 mm (width/length; x/y) for an 8- to 10-band design andadhesively bonded to a backing sheet (for example from G&L). Twoabsorption pads (Millipore) measuring 19×17 mm and overlapping themembrane by 7 mm are adhesively bonded to the ends of the membranedistal to the work zone. 6 mm-long bands (each 0.6 μl) of solutions ofdifferent blood-group-specific antibodies are applied to the indicatorzone regions, so as to be offset in two linear rows, using a dispenser,for example AD3200 (Biodot):

anti-Jka: clone P3HT7 (Diagast, ref. 78003); anti-Jkb: clone P3 143(Diagast, ref. 78004); anti-Fya: clone P3TIM+anti-IgG clone MS278 (MerckMillipore, VL+JZ); anti-Fyb: clone SpA264LBg1 (Merck Millipore, FF);anti-S: clone P3S13JS123+anti-IgG clone MS278 (Diagast, ref. 78007+MerckMillipore, JZ); anti-s: clone P3BER (Merck Millipore, FE); anti-P1:clone P3MON2 (Merck Millipore, VN); anti-k: clone P3A1180L67+anti-IgGclone MS278 (Merck Millipore, FA+JZ). All the antibodies areconcentrated about 10 times before formulation.

The anti-Jka antibody is positioned to the left of the work zone inposition x=3 mm/y=9 mm to y=15. Three other antibodies (anti-Jkb,anti-Fya and anti-Fyb) are dispensed iteratively at intervals of x=2.5mm in parallel with the position of the anti-Jka antibody. The anti-Santibody is positioned to the right of the work zone in position x=3mm/y=34 mm to y=40. Three other antibodies (anti-s, anti-k and anti-P1)are dispensed iteratively at intervals of x=2.5 mm to the position ofthe anti-S antibody.

The anti-erythrocyte-specific validation antibody (Val=process control;rabbit IgG fraction of anti-human RBC, Rockland, 209-4139) is applied asa dot in x=2.5 mm/y=3 mm offset to the last band of the series of theblood-group-specific antibodies. The control dot (Ctl=negative control;contains all the constituents of the various antibody formulations withthe exception of the antibody) is applied in y=3 mm offset to the Valdot. All the antibody solutions contain 1% BSA and 9.4% APP3 solution[32.4% (w/v) D(+)-trehalose dihydrate, 0.055% (v/v) Genapol PF10, 21.8%(v/v) methanol, PPB buffer: 15 mM potassium phosphate buffer/10 mMNaCl/0.05% (w/v) NaN₃]. The antibodies are diluted in 0.07M Tris/HClbuffer, having a pH of 7, with the exception of anti-P1, which isdiluted in 0.01 M citrate buffer, having a pH of 4, as follows: anti-Jka1:5, anti-Jkb 1:5, anti-Fya 1:5+anti-IgG 1:25, anti-S 1:5+1:100, anti-s(small) 1:16.7, anti-k 1:10+anti-IgG 1:100, anti-P1 1:10 and anti-RBC1:10. After the antibodies have been dispensed, the membranes are driedfor 1 hour at 45° C. and welded together with a sealing element in apolycarbonate casing (Medion Grifols Diagnostics AG).

Test Set-Up:

The blood samples can be taken in tubes containing conventionalanticoagulants (for example EDTA, CPDA-1, ACD, citrate) or in nativeform.

In a test tube, 1 drop (50 W) of anticoagulated whole blood is mixedwith 4 drops (200 μl) of Diluent F (Medion Grifols Diagnostics) or 1drop of erythrocyte sediment is mixed with 8 drops (400 μl) of DiluentF, or 2 drops (100 l) of the cells of coagulated blood are mixed with 2drops of Diluent F.

Two drops (100 μl) of the resulting suspension are applied to the workzone of the described test arrangement. After 30 seconds, 6 drops (300μl) of Diluent F are applied to the work zone. After 5 minutes, theresults are read off and recorded.

Result:

The test is valid if the anti-RBC validation dot (val) shows a clearlypositive signal (red dot) and the control dot (ctl) indicates a negativeresult. The presence of a red band indicates that the tested bloodsample is positive for the particular blood group feature. The absenceof a band in the corresponding position in the work zone means that thetested blood sample is negative for the corresponding blood groupfeature.

FIG. 2 shows the successful simultaneous determination of the bloodgroup antigens Jka, Jkb, Fya, Fyb, S, s, k and P1. The donor isJka−Jkb+Fya−Fyb+S−s+k+P1+.

Example 2: Blood Group Determination by Means of the Method According tothe Invention and Comparative Example

The test strip was produced analogously to Example 1. There were used asantibodies: anti-D, clone ESD-1 (Alba), human IgG; anti-k (cellano),clone P3AI 180L67 (Millipore), human IgG; anti-Fya, clone P3TIM(Millipore), human IgG, and as the second antibody: anti-IgG, cloneMS278 (Millipore), mouse IgM.

FIG. 3a shows the dispensing plan used. FIG. 3b to 3e show theexperimental results obtained with samples from different donors. It canclearly be seen that only the determination by means of a first antibodyof the IgG class directed against the blood group antigen and a secondantibody directed against that first antibody leads to a clearlydetectable band, whereas the determination using the first antibody ofthe IgG class does not lead to a clearly recognisable band. On theright-hand side, anti-IgG is applied three times as a further negativecontrol. FIG. 3b to 3e show that no band was obtained in the case ofthis negative control.

What is claimed is:
 1. A device for determining the presence of at leastone blood group antigen in a liquid sample, the device comprising: aseparating matrix comprising tubes filled with gel material, wherein thegel material comprises at least one indicator zone, the indicator zonecomprising: a first antibody, or a fragment thereof, directed againstthe blood group antigen, and a binding element directed against thefirst antibody, the first antibody being an incomplete antibody.
 2. Thedevice of claim 1, wherein the blood group antigen is selected from thegroup consisting of A, B, AB, D, C, E, c, e, Cw, K, k, Jka, Jkb, Fya,Fyb, M, N, S, s, P1, Kpa, Kpb, Lua, Lub, Lea, Leb, Mia, Dia, Jsa, Jsb,Coa, Cob, Wra and Xga.
 3. The device of claim 1, wherein the firstantibody is an antibody of the IgG or IgA type.
 4. The device of claim1, wherein the binding element directed against the first antibody isselected from an antibody directed against the first antibody, or afragment thereof, and a lectin or a fragment thereof.
 5. The device ofclaim 1, wherein the binding element directed against the first antibodyis selected from an anti IgG antibody directed against the firstantibody, or a fragment thereof, an anti-IgA antibody directed againstthe first antibody, or a fragment thereof, protein A, or a fragmentthereof, and protein G, or a fragment thereof.
 6. A method for producingthe device of claim 1, comprising: applying a first antibody, or afragment thereof, directed against the at least one blood group antigenand a binding element directed against the first antibody in theindicator zone, wherein the first antibody is an incomplete antibody. 7.A method for producing the device of claim 1, wherein the first antibodyand the binding element are applied separately from one another or as amixture.
 8. A method for determining at least one blood group antigen ina liquid sample, comprising: i) applying the sample to the device ofclaim 1, wherein said sample is present in an amount sufficient to causethe sample liquid to flow through the indicator zone of the device suchthat the at least one blood group antigen in the sample liquid forms acomplex in the indicator zone, and ii) detecting the formation of thecomplex in the indicator zone.
 9. The method of claim 8, wherein theliquid sample preferably consists of blood or constituents of blood,preferably of whole blood, erythrocyte concentrate, coagulated blood ortest liquid, such as control blood
 10. The method of claim 8, whereinthe sample is diluted with a buffer before it is applied to the device.11. A device for simultaneously determining at least a first and asecond blood group antigens in a liquid sample, comprising: a pluralityof tubes filled with gel material, the gel material comprising at leastone indicator zone, wherein: (i) the at least one indicator zone of thegel material of a first tube of the plurality of tubes comprises: (a) afirst antibody, or a fragment thereof, directed against the first bloodgroup antigen, the first antibody being an incomplete antibody, and (b)a binding element directed against the first antibody; and (ii) the atleast one indicator zone of the gel material of a second tube of theplurality of tubes comprises: (a) a first antibody directed against thesecond blood group antigen, the first antibody being a completeantibody; or (b) a first antibody directed against the second bloodgroup antigen, the first antibody being incomplete, and a bindingelement directed against the first antibody.
 12. The device of claim 11,wherein the first antibody, or a fragment thereof, directed against thefirst blood group antigen is an anti-IgG antibody and the firstantibody, or a fragment thereof, directed against the second blood groupantigen is an anti-IgM antibody.
 13. A method for producing the deviceof claim 11, comprising: applying a first antibody, or a fragmentthereof, directed against the first blood group antigen, and a bindingelement directed against the first antibody in the indicator zone of thefirst tube of the plurality of tubes, wherein the first antibody is anincomplete antibody.
 14. A method for producing the device of claim 11,wherein the first antibody and the binding element in the indicator zoneof the first tube of the plurality of tubes, are applied separately fromone another or as a mixture.